I. Field of the Invention
This invention relates in general to certain new and useful improvements in the stabilization of diagnostic reagent compositions and the method of stabilizing, and, more particularly, to liquid stabilized phosphate-containing substrates in diagnostic reagent compositions and the method of stabilization.
II. Description of the Prior Art
It has recently been estimated that 25% of all in vitro diagnostic tests conducted annually in this country are not reliable. Unreliable tests can result in unnecessary medical treatment, the withholding of necessary treatment and lost income. Because of their high specificity, the use of enzyme determinations has significantly increased during the last few years and indications are that this trend will continue. However, rigorous quality control measures are required to assure the accuracy and consistency of results. This requirement stems from the fact that the exact nature of enzymes, as well as the mechanisms of their reaction, remains unknown for the most part.
At present, the greatest limitation in the diagnostic reagent manufacturer, by far, lies in the unstable characteristics of its product. Current methodologies require the use of numerous labile ingredients, and these ingredients are more likely to increase, rather than decrease, in number. Due to these severe restraints, rigorous quality control is required, and this quality control is, of course, costly. Moreover, if control in any step in the process is not maintained within high degree of control standards, the quality of the final product can be reduced materially.
Several diagnostic reagents containing organic groups and particularly diagnostic reagents containing organic aromatic or cyclic groups, along with hydrolizable phosphate groups, as for example, paranitrophenolphosphate, are used in the determination of various enzymes, such as those enzymes known as acid phosphatase and alkaline phosphatase. These enzymes, and particularly the alkaline phosphatase enzyme, are found in blood serum and are used for detecting abnormal conditions when present in an elevated state. The alkaline phosphatase enzyme is essentially a liver function enzyme, but is also found in bones and in bone portions of the body and in the intestines of human and animal bodies. Increased level of the alkaline phosphatase enzyme is generally indicative of liver malfunction. Acid phosphatase enzyme determination is also highly useful since it is derived from the prostate gland and is used for diagnosis of prostatic cancer.
The phosphatase enzymes are particularly effective for determination inasmuch as they have high affinity for phosphate groups which are attached to organic groups, and particularly to organic cyclic or aromatic groups, and will split the phosphate group from the remaining radical. Accordingly, measurement of alkaline phosphatase and acid phosphatase, for example, is made in the presence of a phospho-organic compound and also in the presence of magnesium ions. Measurement of the phosphate ions produced by reaction with the phosphate enzymes in a given time frame provides a fairly accurate determination.
Several approaches in the determination of the alkaline and acid phosphatase enzymes are commercially employed. In one reaction determination, alkaline phosphatase is determined by means of phenolphosphate in which the phenolphosphate, in presence of magnesium ions, is incubated along with the alkaline phosphatase. The resulting free phenol, due to the alkaline phosphatase activity, is measured after a time period in which the reaction has taken place. In like manner, glycerolphosphate may be used. One very common substrate which is used for determination with the phosphatase enzymes is paranitrophenolphosphate. In this case, a nitro group is attached to the phenol moiety in a para position and a phosphate group is attached to the phenol group by weak oxygen bonding. During the reaction with the alkaline phosphatase, the phosphate group is split, producing free paranitrophenol. The paranitrophenolphosphate is a colorless material at alkaline pH, whereas the nitrophenolphosphate is a yellow colored substance at alkaline pH.
These various substrates which are used in the determination of the alkaline phosphatase and the acid phosphatase are labile organic compounds in that they hydrolize in aqueous solutions and even in alcholic solutions and other organic solutions, thereby dissociating the phosphate group. Thus, substrates are considered labile from the standpoint that the phosphate is hydrolyzed in aqueous solution and hence the substrate does not retain its molecular properties. For this reason, in the present commercialization of alkaline phosphatase reagents and acid phosphatase reagents, a proper buffer is employed to establish the pH. Thus, the phosphate-containing compounds, such as the paranitrophenolphosphate, are provided in the dry state, even in the presence of the magnesium salt providing the magnesium ions. When making a determination, a buffer is added to the dry paranitrophenolphosphate, creating an aqueous solution of the buffer and the paranitrophenolphosphate with this resulting solution used to determine the activity of the phosphatase enzymes.
In accordance with the present invention, it has been found that it is possible to store the paranitrophenolphosphate and similar labile substrates in a liquid media in the presence of stabilizing reagents which effectively stabilize the paranitrophenolphosphate and similar labile components and keeps the phosphate group protected from hydrolysis in the liquid media.
The present commercial state of the art used for stabilizing the reactive ability of the substrates is by locking them into a solid matrix, either by freeze drying, or dry blending such as used for tableting dried powders, primarily in the pharmaceutical diagnostic and related industries and immobilization by locking the chemical structure of the substrate in a solid matrix. Contrary to the sophistication these terms imply, these approaches are neither practical nor desirable and are also expensive. The manufacturer is forced to remove the water and supply a partial product, thus relinquishing part of the quality control cycle in the dilution and use of the final product. Laboratories are forced to pay the high cost of packaging, reagent waste, freeze drying and dry blending, and usefulness of the product is further limited by packaging modes and sizes.
Furthermore, good product uniformity is difficult to achieve. This condition is exemplified by the fact that most commercial freeze dried control sera (reference serum) list the acceptable bottle-to-bottle variation of enzyme constituents at .+-. 10% of the mean.
The present invention is uniquely designed so that the labile ingredients in a liquid reagent solution are effectively "stabilized" by inhibiting hydrolization of the phosphate group forming part of the substrate, thereby controlling the activity of the labile ingredients in a liquid solution against reactivity. This means of stabilization ensures long-term stability in a liquid media. Moreover, close tolerance control can be achieved in the manufacturing of a high quality product which eliminates the inconvenience of the rigid package size and the high cost of packaging and freeze drying and reagent waste.